Systems and methods for whole genome amplification

ABSTRACT

Provided herein are systems and methods for whole genome amplification and sequencing. In particular, provided herein are systems and methods for detection of nucleic acid variants (e.g., rare variants) in limited samples.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application claims priority to U.S. Provisional Application62/118,131, filed Feb. 19, 2015, which is herein incorporated byreference in its entirety.

FIELD OF THE DISCLOSURE

Provided herein are systems and methods for whole genome amplificationand sequencing. In particular, provided herein are systems and methodsfor detection of nucleic acid variants (e.g., rare variants) in limitedsamples.

BACKGROUND

Geneticists are striving to characterize the genetic source of complexdiseases including cancer, autoimmune and neurological disorders.However, the underlying mechanisms driving these diseases remainelusive. It is believed that both germline and somatic mutations incombination with spontaneous variants that accumulate in cells over alifetime, are major factors that drive disease onset and reoccurrence.

Whole genome amplification (WGA) followed by next generation sequencing(NGS) is often utilized to identify nucleic acid mutations. However,existing WGA methods often introduce artifacts and errors, especiallywhen starting with limited amounts of template. Such errors make itdifficult to detect rare mutations among the noise introduced by theamplification and sequencing techniques employed.

As a consequence, a need exists to amplify DNA and RNA from limitedsamples (such a single cells) with high fidelity and low amounts ofTemplate Independent DNA Amplification (TIDA).

SUMMARY

Provided herein are systems and methods for whole genome amplificationand sequencing. In particular, provided herein are systems and methodsfor detection of nucleic acid variants (e.g., rare variants) in limitedsamples (e.g., single cells and/or a limited number of cells).

Embodiments of the present disclosure provide improved methods for wholegenome amplification (WGA) and next generation sequencing (NGS) onlimited sample. The systems and methods described herein eliminatesample clean-up steps and achieve whole genome (optionally enriched fora specific target) amplification with low no-template amplification. Thesystems and methods find use, for example, in detection of nucleic acidvariants in samples of genomic DNA and/or RNA.

For example, in some embodiments, the present disclosure provides amethod of WGA of nucleic acids, comprising: a) contacting a sample ofgenomic DNA with a phi29 polymerase and a heat stable DNA polymerase;and b) amplifying said genomic DNA to generate amplified DNA. In someembodiments, the contacting further comprises contacting with dNTPs,nucleic acid primers, and a buffering agent. In some embodiments, thesample of genomic DNA is a single cell sample. In some embodiments, thesample of genomic DNA is a plurality of cells. In some embodiments, themethod further comprises the step of detecting one or more nucleic acidvariants in said amplified DNA (e.g. including but not limited to,single nucleotide polymorphisms, single nucleotide variations, copynumber variations, gene fusions, nucleic acid insertions, or nucleicacid deletions). In some embodiments, the method further comprises thestep of performing a nucleic acid sequencing assay on said amplified DNA(e.g., a next generation sequencing assay). In some embodiments, theamplification and/or sequencing assays are conducted in a volume ofapproximately 35 to 500 nL. In some embodiments, at least subsets ofsaid primers are specific for a nucleic acid target of interest. In someembodiments, at least a subset of said primers comprises a samplebarcode, molecular barcode, label, or tag sequence. In some embodiments,a no-template control sample does not result in amplified DNA. In someembodiments, the primer comprises a single primer. In some embodiments,the amplification and said sequencing are performed in a single tube. Insome embodiments, the amplification comprises both WGA (e.g., with asingle primer) and PCR (e.g., with a plurality of target specificprimers). In some embodiments, the reaction is performed in amicrofluidic chip comprising a plurality of wells. In some embodiments,each of said wells comprises a distinct nucleic acid primer. In someembodiments, the primers generate amplicons lacking step loopstructures. In some embodiments, at least one primer comprises 3′ endscomprising 3-8 nucleotides designed to bind to intron/exon boundaries ina target nucleic acid. In some embodiments, the nucleotides are, forexample, YAG, YAC, RTC or RTG. In some embodiments, at least one primercomprises 3′ ends comprising 3-8 nucleotides that bind to repeat elementconsensus sequences (e.g., including but not limited to, transposons,retrotransposon, Alu1, 5′ AGCT 3′, LINE, SINE, interspersed repeatelements, cladistic marker sequences or transcription factor bindingsites). In some embodiments, at least one primer comprises 5′ ends thathybridize to a sequencing flow cell.

Further embodiments provide a system or kit, comprising: a) a phi29polymerase; b) a heat stable polymerase; and c) at least one nucleicacid primer (e.g., wherein each of the at least one primers has the sameor different nucleic acid sequences).

Additional embodiments are described herein.

DESCRIPTION OF THE FIGURES

FIG. 1 illustrates a reagent addition protocol displaying volumes andthermal conditions for WGA methods described herein.

FIGS. 2A-2B illustrates the results of amplicons generated from the sameDNA source Nextera “tagmented”, PCR re-amplified and sized using anAgilent Hi sensitivity Bioanalyzer chip (Agilent) using MALBAC (FIG. 2A)and methods of embodiments of the present disclosure (FIG. 2B).

FIGS. 3A-C illustrate observed sequenced counts (Y axis) per chromosome(X axis) and a Pearson coefficient (R²) for MALBAC and method ofembodiments of the present disclosure.

FIGS. 4A-C illustrate observed sequenced counts per chromosome (Y axis).Expected distribution (X axis) and Pearson coefficient R² for MALBAC andmethods of embodiments of the present disclosure.

FIGS. 5A-B illustrate a generalized 53 mer WGA priming sequence bearinginline SBC and MBC. FIG. 5A illustrates those primers of the generalform. FIG. 5B illustrates an exemplary in line dual SBC and MBCprimer-amplicon configuration.

FIG. 6 illustrates in chip WGA Cq and Tm heat maps for 6 and 60 pg ofgDNA positive control (1 and 10 cell equivalents) and individual 1 and10 cell samples as dispensed in the chip for WGA methods of embodimentsof the present disclosure.

FIGS. 7A-B illustrate sequenced bases covered/chromosome/total basescovered for U937 gDNA (positive control).

FIGS. 8A-B illustrates sequenced bases covered/chromosome/total basescovered for and 1 and 10 cell samples respectively.

DETAILED DESCRIPTION

Provided herein are systems and methods for WGA and sequencing. Inparticular, provided herein are systems and methods for detection ofnucleic acid variants (e.g., rare variants) in limited samples (e.g.,single cells or limited numbers of cells).

Provided herein are WGA and sequencing systems and methods for detectingSNP, SNV, rare genetic variants CNV and larger scale geneticrearrangements (e.g., from limited sample amounts). The systems andmethods described herein provide the advantage of analyzing single cellamounts of DNA, performing biochemical reactions (e.g., cell lysis andsample prep), and performing WGA and the Polymerase Chain Reaction (PCR)in small sample volumes (e.g., between 35 and 500 nL, although largervolumes can be utilized).

In some embodiments, the systems and methods utilize phi29 DNApolymerase, a heat stable DNA polymerase, dNTPs, primers, and abuffering agent. This formulation is employed such that only minimalvolume changes and no sample cleanup are utilized. The formulation alsoenables addition of additional oligonucleotides to ameliorate and avoid“allele drop out” phenomena, or to selectively enrich for one or moregiven target regions, using a plurality of one or a pool of primers

In experiments described herein, it was demonstrated that the systemsand methods described herein generated similar data when compared to theMultiple Annealing and Looping Based Amplification Cycles (MALBAC)method (de Bourcy, et al., (2014) PloS one, 9, e105585; Hou, et al.,(2013) Cell, 155, 1492-1506; Huang, et al., (2014) Fertil Steril; Yu, etal., (2014) Anal Chem, 86, 9386-9390; Zong, et al., (2012) Science, 338,1622-1626). The systems and methods described herein have the addedbenefit of (i) employing a higher proofreading polymerase than used inMALBAC; (ii) limits the opportunity for generating undesired recurringDNA rearrangement during amplification, and (iii) decreases theopportunity to generating TIDA. Moreover, the process generates shorteramplicons than typically seen in WGA amplifications, thereby makingthese amplicons more amenable for subsequent PCR amplification and NGSsample preparation methodologies. This provides much needed efficiencyadvantages for high throughput parallel and/or sequencing analysis oflarge number of samples (e.g., 100, 1000, 10,000, or more samples (e.g.of single cells)).

WGA is an in vitro method employed to amplify DNA from limited samplesfor further molecular genetic analyses. Comprehensive human genomeamplification from single cell amounts of DNA is possible. Severalmethods have been developed to amplify whole genomes including PrimerExtension Preamplification (PEP) (Zhang et al., (1992) Proceedings ofthe National Academy of Sciences of the United States of America, 89,5847-5851) and Degenerate Oligonucleotide Primed PCR (DOP-PCR) (Aubele,M. and Smida, J. (2003) Methods in molecular biology, 226, 315-318; Denget al., (2012) Fa Yi Xue Za Zhi, 28, 41-43; Telenius et al., (1992)Genomics, 13, 718-725). Amplification yields, imbalanced amplificationin addition to allele dropout (ADO) associated with these technologieshave limited their broad utilization. The most recent advancement in WGAtechnology is multiple displacement amplification (MDA), also known asstrand displacement amplification. MDA typically employs random hexamerspermitting recurrent polymerase driven strand-displacement synthesis. InMDA the polymerase induces DNA strand displacement “melting” andpolymerization at primer binding sites. As amplicon concentrationsincrease, the number of priming events also increases. The result issimultaneous priming and extension forming an interlacing network ofhyper-branched DNA structures (de Bourcy et al., (2014) PloS one, 9,e105585, Gardner et al., (2014) Adv Bioinformatics, 2014, 101894; Liu etal., (2014) Gene, 550, 131-135; Nelson, J. R. (2014) Curr Protoc MolBiol, 105, Unit 15 13). The reaction can be catalyzed by the phi29 DNApolymerase (Phi29) or by the large fragment of the Bst DNA polymerase(Bst) (Huang et al., (2014) Fertil Steril; Zong et al., Science, 338,1622-1626). MDA type methods sometimes require many hours (e.g., >=4hours) in order to generate a sufficient fold amplification from singlecell amounts of material (Nelson, J. R. (2014), supra). All currentlyavailable WGA techniques have the limitation of generating DNA withincomplete coverage of loci throughout the genome (ADO), particularlywhen little amounts of starting material are used. For example, when asingle cell is used, ADO rates from single-cell WGA, whether by MDA orPCR-based methods, range from 25 to 33% (Spits et al., (2006) Hum Mutat,27, 496-503).

The Bst DNA polymerase (Bst) has recently been championed in anamplification method known as Multiple Annealing and Looping BasedAmplification Cycles (MALBAC) (See e.g., de Bourcy et al., (2014) PloSone, 9, e105585; Hou et al., (2013) Cell, 155, 1492-1506; Huang et al.,(2014) Fertil Steril; Yu et al., (2014) Anal Chem, 86, 9386-9390; Zonget al., (2012) Science, 338, 1622-1626)). Bst, however, has highincorporation error and low processivity rates of 1.5×10⁵ and 15-25 ntrespectively (Aviel-Ronen et al., (2006) BMC genomics, 7, 312; Huang etal., (1999) Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao (Shanghai), 31,379-384; Mead et al., (1991) Biotechniques, 11, 76-78, 80, 82-77).Moreover, Bst lacks a 3′→5′ exonuclease activity (“proof readingactivity”). Phi29 DNA polymerase, on the other hand, possesses highproofreading activity resulting in error rates 10× times lower than Bst,e.g., error and processivity rates of 1-2×10⁻⁶ and 70,000 ntrespectively. Simplistically, these enzymatic measurements would apriori commend the preferential use of the phi29 DNA polymerase for MDAtype applications. However, a fundamental concern for WGA users is thedesire to remove or ameliorate generating artifactual amplicons. Inbrief, low temperature MDA/WGA reactions are thought to lead to largeamounts of TIDA and chimera sequences that do not belong to the originalgenome. These “errors” introduce artifacts into whole genome sequencinganalysis. The present disclosure is not limited to a particularmechanism. Indeed, an understanding of the mechanism is not necessary topractice the disclosure. Nonetheless, it is contemplated that TIDA isthe result of the random hexamer primers becoming templates for phi29polymerase (Holbrook et al., (2005) J Biomol Tech, 16, 125-133). Whileother methods for WGA have been proposed (e.g., PEP and DOP) suchmethods are inefficient, complex and expensive and suffer their own TIDAand ADO errors. Although investigation into the phi29 TIDA phenomenon isgenerally lacking in the literature some authors have investigated phi29incubation at 40° C. rather than at 30° C., phi29's optimum temperature(Alsmadi et al., (2009) BMC Res Notes, 2, 48). Recently, a reportproposing limited Phi29 amplification at 30° C. for strictly 80 min. hasrecently been proposed (Wang et al., (2014) Nature, 512, 155-160) tolimit enzyme “infidelity”.

Based on those concerns, some investigators have resorted to using thelower processivity and non-proof reading enzyme Bst polymerase. This isbecause Bst is capable of amplification at high temperatures (55° C.)and can tolerate limited heat denaturation of DNA templates, whereasphi29 is heat labile. Moreover, some reports have demonstrated improvedgenomic coverage when amplification by Bst polymerase as compared tothat of phi29 (Lage et al., et al. (2003) Genome research, 13, 294-307).However, the Bst TIDA-reduction approach (Lage et al., supra) employednitroindole-modified primers, and the modality of artifact reductionusing those expensive modifications is unknown (Lage et al., supra). Therelative heat tolerance of Bst has also been harnessed using the MALBACprocedure. The MALBAC process employs five cycles of poorly understood“quasi linear” pre-amplification. MALBAC primers containing a 27nucleotide common 5′ sequence region which can loop back on themselvesa, 5 N positions and a 3′ trinucleotide sequence GGG or TTT sequence. Inprinciple, such loops would be expected to display first order-likereaction kinetics rapidly forming panhandle suppression structures(Jones, D. H. (1995) PCR Methods Appl, 4, S195-201; Jones, (1992)Nucleic acids research, 20, 595-600; Jones, D. H. and Winistorfer, S. C.(1993) PCR Methods Appl, 2, 197-203; Park et al., (2003) Biotechniques,34, 750-752, 754-756; Robinson et al. (2006) Genes Chromosomes Cancer,45, 740-753) that prevent additional copying from the initial template.Suppressing amplification from previously amplified fragments isbelieved to reduce the amplification bias commonly associated with theuneven exponential amplification of DNA fragments by typical WGA and PCRmethods (Holbrook et al., (2005) J Biomol Tech, 16, 125-133; WO2012166425).

Despite this conceptual but undemonstrated panhandle suppressiontechnique claimed in MALBAC, it has remained difficult to genericallyemploy the MALBAC single cell technique in situations requiring bothhigh amplicon-template fidelity and ease of use in microfluidicdispenses and devices. The major reasons for not employing “Bst-basedMALBAC” are intrinsic to the polymerase incorporation error rates whichinclude the facts that stochastic-like dNTP incorporation errors in thefirst cycle of MALBAC are propagated throughout subsequent amplificationsteps. As a consequence, MALBAC data requires comparing single cellsequencing results to those obtained from 2-3 cells within the samelineage, as well as to cells from an unrelated lineage to obtainsequencing “truth”. Secondly, genome coverage at a single cell levelusing MALBAC is less uniform than bulk sequencing. MALBAC it is unableto detect approximately one third of SNPs compared to bulk sequencing(de Bourcy et al., (2014) PloS one, 9, e105585; Hou et al., (2013) Cell,155, 1492-1506; Huang et al., (2014) Fertil Steril; Yu et al., (2014)Anal Chem, 86, 9386-9390; Zong et al., (2012) Science, 338, 1622-1626).Thirdly, in common with typical MDA procedures, MALBAC employs differingvolumes of reagents for sample lysis, MDA and PCR. This renders MALBACautomation difficult for scale up when examining large numbers of singlecell samples in simple microfluidic systems. Fourthly, in pre-kittedformulations, the addition of specific primers into the MDA reaction mixwith the aim to amplify underrepresented genomic areas (e.g., ADOregions) or enrich for specific target sequences is not easily performed(US Pat. App. No. 20120100549 and WO 2008051928).

Investigations of varying commercial suppliers of MDA-like systems knownas PicoPLEX (Rubicon Genomics), MALBAC, (Yikon Genomics), RepliG(Qiagen) Illustra GenomiPhi (GE Health Care) reveal those kits areinflexible regarding primer(s), enzyme(s) and do not permitcustomization/scalability for microfluidic use. Moreover, thoseformulations are typically “company secrets”, constraining users tousing predefined fixed volumes and concentrations. This renderstranslation of commercial kit protocols impractical or impossible inmany conventional and microfluidic biochemical configurations.Therefore, a need exists for alternate MDA methods of amplifying smallamounts of genomic DNA, such as from a single cell or small group ofcells and limited samples in general with a strong proof readingpolymerases that nevertheless generate limited amounts of TIDA.

There is generally a great need in the biological sciences for arepresentative, unbiased WGA method that enables: i) high polymeraseprocessivity, strand displacement and 3′-5′ “proof reading”; ii) reducedchimera and TIDA generation, iii) increased coverage/decreased ADO; iv)permits selective target enrichment; and v) permits simplified liquidhandling and reagent automation in microfluidic sample preparationmethodologies. More specifically, there is a great need for simplifiedand robust methods and kits that allow for the robust amplification ofnucleic acid molecules from limited samples inputs including single cellsamples. There is also a need for improved methods, compositions,systems, apparatuses and kits that allow enrichment for selectiveamplification of nucleic acid molecules from low input nucleic acidsamples, and tagging each primer while avoiding, or minimizing, theformation of artifacts. More specifically, there is a need in the artfor improved methods, compositions, systems and kits that allow for thesimultaneous specific target enrichment within the MDA/WGA reaction oftens, hundreds and thousands of target-specific nucleic acid moleculesin a single reaction vessel which can be used for any applicabledownstream assay or analysis.

The formulation utilized in the compositions and methods describedherein overcome many of those difficulties and generates data superiorto MALBAC, employs a higher proofreading polymerase and a simplifiedprocess. In particular, the systems and methods described herein aboveavoid the artifacts and TIDA commonly observed with MALBAC and other WGAmethods and provide the further advantage of the option of specificallytargeting genomic regions of interest.

In some embodiments, the systems and methods described herein employ thesame primer design strategies described in the MALBAC protocol toameliorate potential inter and intra self-primer generation (Holbrook etal., (2005) J Biomol Tech, 16, 125-133). Uniquely, this MALBAC-styledreaction was refashioned to work using the phi29 polymerase instead ofthe Bst polymerase for enzyme biochemistry template fidelity reasonsdescribed above. The phi29 reaction temperatures and time were furtheraltered so that TIDA was eliminated and NTCs were reproducibly negative(Alsmadi et al., (2009) BMC Res Notes, 2, 48) as judged by bioanalyzertraces. In conclusion, the MDA amplification procedure has been alteredto enable use of the high fidelity and high processivity of phi29polymerase resulting in a formulation that is easy to use formicrofluidic and robotics platforms.

In some embodiments, the reaction mixture is supplemented with specificprimers to enrich for specific targets as per user needs. In someembodiments, primer sequences are modified to contain tags that enablemolecular and sample barcoding and permit use with instrument NGS flowcells.

The methods disclosed herein for WGA and optionally target-specificamplification may comprise conducting one or more amplificationreactions. Conducting one or more amplification reactions may compriseone or more PCR-based amplifications, non-PCR based amplifications, or acombination thereof. Illustrative non-limiting examples of nucleic acidamplification techniques include, but are not limited to, polymerasechain reaction (PCR), reverse transcription polymerase chain reaction(RT-PCR), nested PCR, linear amplification, multiple displacementamplification (MDA), real-time SDA, rolling circle amplification,circle-to-circle amplification transcription-mediated amplification(TMA), ligase chain reaction (LCR), strand displacement amplification(SDA), and nucleic acid sequence based amplification (NASBA). Those ofordinary skill in the art will recognize that certain amplificationtechniques (e.g., PCR) require that RNA be reversed transcribed to DNAprior to amplification (e.g., RT-PCR), whereas other amplificationtechniques directly amplify RNA (e.g., TMA and NASBA).

The polymerase chain reaction (U.S. Pat. Nos. 4,683,195, 4,683,202,4,800,159 and 4,965,188, each of which is herein incorporated byreference in its entirety), commonly referred to as PCR, uses multiplecycles of denaturation, annealing of primer pairs to opposite strands,and primer extension to permit exponential increase in copy numbers oftarget nucleic acids. In a variation called RT-PCR, reversetranscriptase (RT) is used to make a complementary DNA (cDNA) from RNA,and the cDNA is then amplified by PCR to produce multiple copies of DNA.For other various permutations of PCR see, e.g., U.S. Pat. Nos.4,683,195, 4,683,202 and 4,800,159; Mullis et al., Meth. Enzymol. 155:335 (1987); and, Murakawa et al., DNA 7: 287 (1988), each of which isherein incorporated by reference in its entirety.

Transcription mediated amplification (U.S. Pat. Nos. 5,480,784 and5,399,491, each of which is herein incorporated by reference in itsentirety), commonly referred to as TMA, synthesizes multiple copies of atarget nucleic acid sequence autocatalytically under conditions ofsubstantially constant temperature, ionic strength, and pH in whichmultiple RNA copies of the target sequence autocatalytically generateadditional copies. See, e.g., U.S. Pat. Nos. 5,399,491 and 5,824,518,each of which is herein incorporated by reference in its entirety. In avariation described in U.S. Publ. No. 20060046265 (herein incorporatedby reference in its entirety), TMA optionally incorporates the use ofblocking moieties, terminating moieties, and other modifying moieties toimprove TMA process sensitivity and accuracy.

The ligase chain reaction (Weiss, R., Science 254: 1292 (1991), hereinincorporated by reference in its entirety), commonly referred to as LCR,uses two sets of complementary DNA oligonucleotides that hybridize toadjacent regions of the target nucleic acid. The DNA oligonucleotidesare covalently linked by a DNA ligase in repeated cycles of thermaldenaturation, hybridization and ligation to produce a detectabledouble-stranded ligated oligonucleotide product.

Strand displacement amplification (Walker, G. et al., Proc. Natl. Acad.Sci. USA 89: 392-396 (1992); U.S. Pat. Nos. 5,270,184 and 5,455,166,each of which is herein incorporated by reference in its entirety),commonly referred to as SDA, uses cycles of annealing pairs of primersequences to opposite strands of a target sequence, primer extension inthe presence of a dNTPαS to produce a duplex hemi-phosphorothioatedprimer extension product, endonuclease-mediated nicking of ahemi-modified restriction endonuclease recognition site, andpolymerase-mediated primer extension from the 3′ end of the nick todisplace an existing strand and produce a strand for the next round ofprimer annealing, nicking and strand displacement, resulting ingeometric amplification of product. Thermophilic SDA (tSDA) usesthermophilic endonucleases and polymerases at higher temperatures inessentially the same method (EP Pat. No. 0 684 315).

Other amplification methods include, for example: nucleic acid sequencebased amplification (U.S. Pat. No. 5,130,238, herein incorporated byreference in its entirety), commonly referred to as NASBA; one that usesan RNA replicase to amplify the probe molecule itself (Lizardi et al.,BioTechnol. 6: 1197 (1988), herein incorporated by reference in itsentirety), commonly referred to as Qβ replicase; a transcription basedamplification method (Kwoh et al., Proc. Natl. Acad. Sci. USA 86:1173(1989)); and, self-sustained sequence replication (Guatelli et al.,Proc. Natl. Acad. Sci. USA 87: 1874 (1990), each of which is hereinincorporated by reference in its entirety). For further discussion ofknown amplification methods see Persing, David H., “In Vitro NucleicAcid Amplification Techniques” in Diagnostic Medical Microbiology:Principles and Applications (Persing et al., Eds.), pp. 51-87 (AmericanSociety for Microbiology, Washington, D.C. (1993)).

In some embodiments, nucleic acid sequencing methods are utilized (e.g.,for detection of amplified nucleic acids). In some embodiments, thetechnology provided herein finds use in a Second Generation (a.k.a. NextGeneration or Next-Gen), Third Generation (a.k.a. Next-Next-Gen), orFourth Generation (a.k.a. N3-Gen) sequencing technology including, butnot limited to, pyrosequencing, sequencing-by-ligation, single moleculesequencing, sequence-by-synthesis (SBS), semiconductor sequencing,massive parallel clonal, massive parallel single molecule SBS, massiveparallel single molecule real-time, massive parallel single moleculereal-time nanopore technology, etc. Morozova and Marra provide a reviewof some such technologies in Genomics, 92: 255 (2008), hereinincorporated by reference in its entirety. Those of ordinary skill inthe art will recognize that because RNA is less stable in the cell andmore prone to nuclease attack experimentally RNA is usually reversetranscribed to DNA before sequencing. A number of DNA sequencingtechniques are suitable, including fluorescence-based sequencingmethodologies (See, e.g., Birren et al., Genome Analysis: Analyzing DNA,1, Cold Spring Harbor, N.Y.; herein incorporated by reference in itsentirety). In some embodiments, the technology finds use in automatedsequencing techniques understood in that art. In some embodiments, thepresent technology finds use in parallel sequencing of partitionedamplicons (PCT Publication No: WO2006084132 to Kevin McKernan et al.,herein incorporated by reference in its entirety). In some embodiments,the technology finds use in DNA sequencing by parallel oligonucleotideextension (See, e.g., U.S. Pat. No. 5,750,341 to Macevicz et al., andU.S. Pat. No. 6,306,597 to Macevicz et al., both of which are hereinincorporated by reference in their entireties). Additional examples ofsequencing techniques in which the technology finds use include theChurch polony technology (Mitra et al., 2003, Analytical Biochemistry320, 55-65; Shendure et al., 2005 Science 309, 1728-1732; U.S. Pat. No.6,432,360, U.S. Pat. No. 6,485,944, U.S. Pat. No. 6,511,803; hereinincorporated by reference in their entireties), the 454 picotiterpyrosequencing technology (Margulies et al., 2005 Nature 437, 376-380;US 20050130173; herein incorporated by reference in their entireties),the Solexa single base addition technology (Bennett et al., 2005,Pharmacogenomics, 6, 373-382; U.S. Pat. No. 6,787,308; U.S. Pat. No.6,833,246; herein incorporated by reference in their entireties), theLynx massively parallel signature sequencing technology (Brenner et al.(2000). Nat. Biotechnol. 18:630-634; U.S. Pat. No. 5,695,934; U.S. Pat.No. 5,714,330; herein incorporated by reference in their entireties),and the Adessi PCR colony technology (Adessi et al. (2000). Nucleic AcidRes. 28, E87; WO 00018957; herein incorporated by reference in itsentirety).

Next-generation sequencing (NGS) methods share the common feature ofmassively parallel, high-throughput strategies, with the goal of lowercosts in comparison to older sequencing methods (see, e.g., Voelkerdinget al., Clinical Chem., 55: 641-658, 2009; MacLean et al., Nature Rev.Microbiol., 7: 287-296; each herein incorporated by reference in theirentirety). NGS methods can be broadly divided into those that typicallyuse template amplification and those that do not.Amplification-requiring methods include pyrosequencing commercialized byRoche as the 454 technology platforms (e.g., GS 20 and GS FLX), LifeTechnologies/Ion Torrent, the Solexa platform commercialized byIllumina, GnuBio, and the Supported Oligonucleotide Ligation andDetection (SOLiD) platform commercialized by Applied Biosystems.Non-amplification approaches, also known as single-molecule sequencing,are exemplified by the HeliScope platform commercialized by HelicosBioSciences, and emerging platforms commercialized by VisiGen, OxfordNanopore Technologies Ltd., and Pacific Biosciences, respectively.

In pyrosequencing (Voelkerding et al., Clinical Chem., 55: 641-658,2009; MacLean et al., Nature Rev. Microbiol., 7: 287-296; U.S. Pat. No.6,210,891; U.S. Pat. No. 6,258,568; each herein incorporated byreference in its entirety), template DNA is fragmented, end-repaired,ligated to adaptors, and clonally amplified in-situ by capturing singletemplate molecules with beads bearing oligonucleotides complementary tothe adaptors. Each bead bearing a single template type iscompartmentalized into a water-in-oil microvesicle, and the template isclonally amplified using a technique referred to as emulsion PCR. Theemulsion is disrupted after amplification and beads are deposited intoindividual wells of a picotitre plate functioning as a flow cell duringthe sequencing reactions. Ordered, iterative introduction of each of thefour dNTP reagents occurs in the flow cell in the presence of sequencingenzymes and luminescent reporter such as luciferase. In the event thatan appropriate dNTP is added to the 3′ end of the sequencing primer, theresulting production of ATP causes a burst of luminescence within thewell, which is recorded using a CCD camera. It is possible to achieveread lengths greater than or equal to 400 bases, and 10⁶ sequence readscan be achieved, resulting in up to 500 million base pairs (Mb) ofsequence.

In the Solexa/Illumina platform (Voelkerding et al., Clinical Chem., 55:641-658, 2009; MacLean et al., Nature Rev. Microbiol., 7: 287-296; U.S.Pat. No. 6,833,246; U.S. Pat. No. 7,115,400; U.S. Pat. No. 6,969,488;each herein incorporated by reference in its entirety), sequencing dataare produced in the form of shorter-length reads. In this method,single-stranded fragmented DNA is end-repaired to generate5′-phosphorylated blunt ends, followed by Klenow-mediated addition of asingle A base to the 3′ end of the fragments. A-addition facilitatesaddition of T-overhang adaptor oligonucleotides, which are subsequentlyused to capture the template-adaptor molecules on the surface of a flowcell that is studded with oligonucleotide anchors. The anchor is used asa PCR primer, but because of the length of the template and itsproximity to other nearby anchor oligonucleotides, extension by PCRresults in the “arching over” of the molecule to hybridize with anadjacent anchor oligonucleotide to form a bridge structure on thesurface of the flow cell. These loops of DNA are denatured and cleaved.Forward strands are then sequenced with reversible dye terminators. Thesequence of incorporated nucleotides is determined by detection ofpost-incorporation fluorescence, with each fluor and block removed priorto the next cycle of dNTP addition. Sequence read length ranges from 36nucleotides to over 250 nucleotides, with overall output exceeding 1billion nucleotide pairs per analytical run.

Sequencing nucleic acid molecules using SOLiD technology (Voelkerding etal., Clinical Chem., 55: 641-658, 2009; MacLean et al., Nature Rev.Microbiol., 7: 287-296; U.S. Pat. No. 5,912,148; U.S. Pat. No.6,130,073; each herein incorporated by reference in their entirety) alsoinvolves fragmentation of the template, ligation to oligonucleotideadaptors, attachment to beads, and clonal amplification by emulsion PCR.Following this, beads bearing template are immobilized on a derivatizedsurface of a glass flow-cell, and a primer complementary to the adaptoroligonucleotide is annealed. However, rather than utilizing this primerfor 3′ extension, it is instead used to provide a 5′ phosphate group forligation to interrogation probes containing two probe-specific basesfollowed by 6 degenerate bases and one of four fluorescent labels. Inthe SOLiD system, interrogation probes have 16 possible combinations ofthe two bases at the 3′ end of each probe, and one of four fluors at the5′ end. Fluor color, and thus identity of each probe, corresponds tospecific color-space coding schemes. Multiple rounds (usually 7) ofprobe annealing, ligation, and fluor detection are followed bydenaturation, and then a second round of sequencing using a primer thatis offset by one base relative to the initial primer. In this manner,the template sequence can be computationally re-constructed, andtemplate bases are interrogated twice, resulting in increased accuracy.Sequence read length averages 35 nucleotides, and overall output exceeds4 billion bases per sequencing run.

In certain embodiments, the technology finds use in nanopore sequencing(see, e.g., Astier et al., J. Am. Chem. Soc. 2006 Feb. 8;128(5):1705-10, herein incorporated by reference). The theory behindnanopore sequencing has to do with what occurs when a nanopore isimmersed in a conducting fluid and a potential (voltage) is appliedacross it. Under these conditions a slight electric current due toconduction of ions through the nanopore can be observed, and the amountof current is exceedingly sensitive to the size of the nanopore. As eachbase of a nucleic acid passes through the nanopore, this causes a changein the magnitude of the current through the nanopore that is distinctfor each of the four bases, thereby allowing the sequence of the DNAmolecule to be determined.

In certain embodiments, the technology finds use in HeliScope by HelicosBioSciences (Voelkerding et al., Clinical Chem., 55: 641-658, 2009;MacLean et al., Nature Rev. Microbiol., 7: 287-296; U.S. Pat. No.7,169,560; U.S. Pat. No. 7,282,337; U.S. Pat. No. 7,482,120; U.S. Pat.No. 7,501,245; U.S. Pat. No. 6,818,395; U.S. Pat. No. 6,911,345; U.S.Pat. No. 7,501,245; each herein incorporated by reference in theirentirety). Template DNA is fragmented and polyadenylated at the 3′ end,with the final adenosine bearing a fluorescent label. Denaturedpolyadenylated template fragments are ligated to poly(dT)oligonucleotides on the surface of a flow cell. Initial physicallocations of captured template molecules are recorded by a CCD camera,and then label is cleaved and washed away. Sequencing is achieved byaddition of polymerase and serial addition of fluorescently-labeled dNTPreagents. Incorporation events result in fluor signal corresponding tothe dNTP, and signal is captured by a CCD camera before each round ofdNTP addition. Sequence read length ranges from 25-50 nucleotides, withoverall output exceeding 1 billion nucleotide pairs per analytical run.

The Ion Torrent technology is a method of DNA sequencing based on thedetection of hydrogen ions that are released during the polymerizationof DNA (see, e.g., Science 327(5970): 1190 (2010); U.S. Pat. Appl. Pub.Nos. 20090026082, 20090127589, 20100301398, 20100197507, 20100188073,and 20100137143, incorporated by reference in their entireties for allpurposes). A microwell contains a template DNA strand to be sequenced.Beneath the layer of microwells is a hypersensitive ISFET ion sensor.All layers are contained within a CMOS semiconductor chip, similar tothat used in the electronics industry. When a dNTP is incorporated intothe growing complementary strand a hydrogen ion is released, whichtriggers a hypersensitive ion sensor. If homopolymer repeats are presentin the template sequence, multiple dNTP molecules will be incorporatedin a single cycle. This leads to a corresponding number of releasedhydrogens and a proportionally higher electronic signal. This technologydiffers from other sequencing technologies in that no modifiednucleotides or optics is used. The per-base accuracy of the Ion Torrentsequencer is ˜99.6% for 50 base reads, with ˜100 Mb to 100 Gb generatedper run. The read-length is 100-300 base pairs. The accuracy forhomopolymer repeats of 5 repeats in length is ˜98%. The benefits of ionsemiconductor sequencing are rapid sequencing speed and low upfront andoperating costs.

The technology finds use in another nucleic acid sequencing approachdeveloped by Stratos Genomics, Inc. and involves the use of Xpandomers.This sequencing process typically includes providing a daughter strandproduced by a template-directed synthesis. The daughter strand generallyincludes a plurality of subunits coupled in a sequence corresponding toa contiguous nucleotide sequence of all or a portion of a target nucleicacid in which the individual subunits comprise a tether, at least oneprobe or nucleobase residue, and at least one selectively cleavablebond. The selectively cleavable bond(s) is/are cleaved to yield anXpandomer of a length longer than the plurality of the subunits of thedaughter strand. The Xpandomer typically includes the tethers andreporter elements for parsing genetic information in a sequencecorresponding to the contiguous nucleotide sequence of all or a portionof the target nucleic acid. Reporter elements of the Xpandomer are thendetected. Additional details relating to Xpandomer-based approaches aredescribed in, for example, U.S. Pat. Pub No. 20090035777, entitled “HighThroughput Nucleic Acid Sequencing by Expansion,” filed Jun. 19, 2008,which is incorporated herein in its entirety.

Other emerging single molecule sequencing methods include real-timesequencing by synthesis using a VisiGen platform (Voelkerding et al.,Clinical Chem., 55: 641-58, 2009; U.S. Pat. No. 7,329,492; U.S. patentapplication Ser. No. 11/671,956; U.S. patent application Ser. No.11/781,166; each herein incorporated by reference in their entirety) inwhich immobilized, primed DNA template is subjected to strand extensionusing a fluorescently-modified polymerase and florescent acceptormolecules, resulting in detectable fluorescence resonance energytransfer (FRET) upon nucleotide addition.

In some embodiments, the systems and methods described herein areutilized in microfluidic systems. For example, in some embodiments, theSmartChip TE available from WaferGen (Fremont, Calif.) (See e.g., U.S.Pat. Nos. 7,833,709, 7,311,794, 7,622,296; herein incorporated byreference in their entirety) are utilized. Such microfluidic systemsprovide a plurality of reaction wells that allow high-throughputnanoliter volume reactions (e.g., from 10 to 300 nL (e.g., 10, 15, 20,25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150,200, 250, 300, 400, or 500 nL, although other volumes are specificallycontemplated)).

In some embodiments, the present disclosure provides kits, systems, andsoftware (e.g., for performing and analyzing WGA assays and/or otherassays). In some embodiments, kits, and systems comprise one or more(e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) nucleic acid primers,primer pairs, or probes). In some embodiments, kits comprise one or moreof buffers, controls, dNTPs, etc. In some embodiments, kits comprisephi29 polymerase and no additional polymerases. In some embodiments,kits comprise phi29 and an additional polymerase (e.g., Bst).

In some embodiments, reaction mixtures comprising a complex of a targetnucleic acid, a primer and phi29 polymerase (e.g., alone or incombination with an additional polymerase) are provided. In someembodiments, reaction mixtures comprise a target or amplicon with one ortwo nucleic acid primers hybridized thereto.

Nucleic acids may also be provided on a solid support. The solid supportmay comprise one or more beads, plates, solid surfaces, wells, chips, ora combination thereof. The beads may be magnetic, antibody coated,protein A crosslinked, protein G crosslinked, streptavidin coated,oligonucleotide conjugated, silica coated, or a combination thereof.Examples of beads include, but are not limited to, Ampure beads, AMPureXP beads, streptavidin beads, agarose beads, magnetic beads, DNYDABEADS,MACS microbeads, antibody conjugated beads (e.g., anti-immunoglobulinmicrobead), protein A conjugated beads, protein G conjugated beads,protein A/G conjugated beads, protein L conjugated beads, oligo-dTconjugated beads, silica beads, silica-like beads, anti-biotinmicrobead, anti-fluorochrome microbead, and BCMAG Carboxy-TerminatedMagnetic Beads.

The compositions and kits may comprise primers and primer pairs capableof amplifying target molecules, or fragments or subsequences orcomplements thereof. The nucleotide sequences of the target moleculesmay be provided in computer-readable media for in silico applicationsand as a basis for the design of appropriate primers for amplificationof one or more target molecules.

Primers based on the nucleotide sequences of target molecules can bedesigned for use in amplification of the target molecules. For use inamplification reactions such as WGA or PCR, a single primer or pair ofprimers can be used. The exact composition of the primer sequences isnot critical to the disclosure, but for most applications the primersmay hybridize to specific sequences of the target molecules or universaltargets under stringent conditions, particularly under conditions ofhigh stringency, as known in the art. The pairs of primers are usuallychosen so as to generate an amplification product of at least about 15or more, 20 or more, 30 or more, 40 or more, 50 or more, 60 or more, 70or more, 80 or more, 90 or more, 100 or more, 125 or more, 150 or more,175 or more, 200 or more, 250 or more, 300 or more, 350 or more, 400 ormore, 450 or more, 500 or more, 600 or more, 700 or more, 800 or more,900 or more, or 1000 or more nucleotides. Algorithms for the selectionof primer sequences are generally known, and are available in commercialsoftware packages. These primers may be used in standard quantitative orqualitative PCR-based assays to assess transcript expression levels oftarget molecules. Alternatively, these primers may be used incombination with probes, such as molecular beacons in amplificationsusing real-time PCR.

One skilled in the art also appreciates that the nucleotide sequence ofthe entire length of the primer does not need to be derived from thetarget sequence. Thus, for example, the primer may comprise nucleotidesequences at the 5′ and/or 3′ termini that are not derived from thetarget molecule. Nucleotide sequences which are not derived from thenucleotide sequence of the target molecule may provide additionalfunctionality to the primer. For example, they may provide a restrictionenzyme recognition sequence or a “tag” that facilitates detection,isolation, purification or immobilization onto a solid support.Alternatively, the additional nucleotides may provide aself-complementary sequence that allows the primer to adopt a hairpinconfiguration. Such configurations may be necessary for certain primers,for example, molecular beacon and Scorpion primers, which can be used insolution hybridization techniques.

The probes or primers can incorporate moieties useful in detection,isolation, purification, or immobilization, if desired. Such moietiesare well-known in the art (see, for example, Ausubel et al., (1997 &updates) Current Protocols in Molecular Biology, Wiley & Sons, New York)and are chosen such that the ability of the probe to hybridize with itstarget molecule is not affected.

Examples of suitable moieties are bar codes (see above), detectablelabels, such as radioisotopes, fluorophores, chemiluminophores, enzymes,colloidal particles, and fluorescent microparticles, as well asantigens, antibodies, haptens, avidin/streptavidin, biotin, haptens,enzyme cofactors/substrates, enzymes, and the like.

A label can optionally be attached to or incorporated into a probe orprimer to allow detection and/or quantitation of a target polynucleotiderepresenting the target molecule of interest. The target polynucleotidemay be the expressed target molecule RNA itself, a cDNA copy thereof, oran amplification product derived therefrom, and may be the positive ornegative strand, so long as it can be specifically detected in the assaybeing used. Similarly, an antibody may be labeled.

In certain multiplex formats, labels used for detecting different targetmolecules may be distinguishable. The label can be attached directly(e.g., via covalent linkage) or indirectly, e.g., via a bridgingmolecule or series of molecules (e.g., a molecule or complex that canbind to an assay component, or via members of a binding pair that can beincorporated into assay components, e.g. biotin-avidin or streptavidin).Many labels are commercially available in activated forms which canreadily be used for such conjugation (for example through amineacylation), or labels may be attached through known or determinableconjugation schemes, many of which are known in the art.

Labels useful in the disclosure described herein include any substancewhich can be detected when bound to or incorporated into the targetmolecule. Any effective detection method can be used, including optical,spectroscopic, electrical, piezoelectrical, magnetic, Raman scattering,surface plasmon resonance, colorimetric, calorimetric, etc. A label istypically selected from a chromophore, a lumiphore, a fluorophore, onemember of a quenching system, a chromogen, a hapten, an antigen, amagnetic particle, a material exhibiting nonlinear optics, asemiconductor nanocrystal, a metal nanoparticle, an enzyme, an antibodyor binding portion or equivalent thereof, an aptamer, and one member ofa binding pair, and combinations thereof. Quenching schemes may be used,wherein a quencher and a fluorophore as members of a quenching pair maybe used on a probe, such that a change in optical parameters occurs uponbinding to the target introduce or quench the signal from thefluorophore. One example of such a system is a molecular beacon.Suitable quencher/fluorophore systems are known in the art. The labelmay be bound through a variety of intermediate linkages. For example, atarget polynucleotide may comprise a biotin-binding species, and anoptically detectable label may be conjugated to biotin and then bound tothe labeled target polynucleotide. Similarly, a polynucleotide sensormay comprise an immunological species such as an antibody or fragment,and a secondary antibody containing an optically detectable label may beadded.

Chromophores useful in the methods described herein include anysubstance which can absorb energy and emit light. For multiplexedassays, a plurality of different signaling chromophores can be used withdetectably different emission spectra. The chromophore can be alumophore or a fluorophore. Typical fluorophores include fluorescentdyes, semiconductor nanocrystals, lanthanide chelates,polynucleotide-specific dyes and green fluorescent protein.

Coding schemes may optionally be used, comprising encoded particlesand/or encoded tags associated with different polynucleotides of thedisclosure. A variety of different coding schemes are known in the art,including fluorophores, including SCNCs, deposited metals, and RF tags.

Polynucleotides from the described target molecules may be employed asprobes for detecting target molecules expression, for ligationamplification schemes, or may be used as primers for amplificationschemes of all or a portion of a target molecule. When amplified, eitherstrand produced by amplification may be provided in purified and/orisolated form.

In some embodiments, at least subsets of primers for WGA comprise amolecular bar code to track individual samples within a pool (e.g., asequencing pool). NGS methods can produce millions even billions ofreads (Metzker, M. L. (2010) Nature reviews. Genetics, 11, 31-46).Nevertheless, due to the inherent noisiness and low fidelity in a NGSsingle read, 20× to 100× read coverage of the same sequence is sometimesutilized to ensure the determined sequence is not erroneous. Methodsknown as “sample barcoding” (SBC) exist to pool multiple, uniquelyidentifiable sample sets colloquially known as “libraries”. SBClibraries can de-multiplexed by post-image-processing bioinformaticanalysis, markedly lowering costs of acquiring datasets for lowcomplexity samples. In practice barcodes consist of DNA sequenceidentifiers that are appended to the ends of DNA fragments (by ligationor inclusion within an oligo by PCR). Binning reads containing the sameSBC was first reported on the Roche 454 sequencing (Kasschau et al.,(2007) PLoS biology, 5, e57) then the Illumina platform (Binladen etal., (2007) PloS one, 2, e197). As sequencing yields have risen, thedegree of multiplexing has also risen, from 96 barcodes (Kozarewa, I.and Turner, D. J. (2011) Methods in molecular biology, 733, 279-298), to2167 barcodes (Caporaso et al., (2012) The ISME journal, 6, 1621-1624)and more recently the software tool TagGD can design up to 20,000-plexbarcode sets (Costea et al., (2013) PloS one, 8, e57521) for theIllumina system. In the Illumina flow cell jargon, barcodes aredescribed as “in line” if they are adjacent to the sample DNA and readfrom the same sequencing primer as part of the sequence read. In lineSBC are obligatory to be read and “take up/consume” some of thesequencing read length (Kircher et al., (2012) Nucleic acids research,40, e3; Indexing and Barcoding for Illumina NextGen Sequencing (2011)University of Massachusetts Medical School).

PCR processes are known to generate chimeras by recombining differenttemplates molecules “a.k.a. jumping PCR” (Fang et al., (1998) Journal ofvirological methods, 76, 139-148; Meyerhans et al., (1990) DNArecombination during PCR. Nucleic acids research, 18, 1687-1691). As aresult, pooled amplicon amplification methods can inadvertentlyintroduce significant levels of sample cross-contamination (˜0.3% i.e. 3in 1000 errors) when sequencing bulk multiplex libraries. On theIllumina platform a double-indexing method, e.g., placing indexes intoboth of the universal adapter sequences can overcome a major errorsource (Kircher et al., supra) i.e. mixed cluster errors on the Illuminaflow cell, Other error sources not necessarily corrected by doubleindexing include sporadic cross contamination introduced during oligosynthesis, sample handling missteps and some jumping PCR events. Methodssuch as utilizing duplex sequencing “molecular barcodes” (MBC) (Schmittet al., (2012) Proceedings of the National Academy of Sciences of theUnited States of America, 109, 14508-14513) or unique identifiers suchas “Safe-SeqS” are employed (Quail et al., (2014) BMC genomics, 15, 110;Kinde et al., (2011) Proceedings of the National Academy of Sciences ofthe United States of America, 108, 9530-9535).

Estimates of “true mutation frequencies” in normal cells generally rangefrom 10-8 to 10-11 per position per haploid genome (Cervantes et al.,(2002) Proceedings of the National Academy of Sciences of the UnitedStates of America, 99, 3586-3590; Roach et al., (2010) Science, 328,636-639). Depending on the read quality required such as looking forrare somatic mutations, (needle in a haystack type applications) dualindexing alone may not provide a sufficient error correctionmethodology. Even when applying high-stringency base calling,conventional analysis using the Illumina platform has an error rate of˜9.1×10-6 errors/bp/PCR cycle. The platform error rate is more than anorder of magnitude higher than the error rate reported for the PhusionDNA polymerase (NEB), a high fidelity polymerase commonly used inlibrary construction. Random errors are also introduced duringoligonucleotide synthesis, whose error rate alone is estimated at 60×more errors than obtained using Phusion PCR (Schmitt et al., (2012)Proceedings of the National Academy of Sciences of the United States ofAmerica, 109, 14508-14513). In aggregate, this indicates that mutationalloads for “rare variants” using conventional NGS analysis are vastlyoverestimated. Consequently, NGS cannot generally be used to detect“rare variants” without error correction due to high NGS error ratesfrom single reads. MBC methods employing technical replicates as a meansof reducing errors have been proposed (Robasky et al., (2014) Naturereviews. Genetics, 15, 56-62). Those approaches propose to ligate/add a12 nucleotide random tag sequence within the body of the in linesequencing primer (Quail et al., (2014) BMC genomics, 15, 110; Kinde etal., (2011) Proceedings of the National Academy of Sciences of theUnited States of America, 108, 9530-9535). Given that each end of thesample molecule can receive a randomized 12-mer tag, if a sufficientlylarge oligonucleotide synthesis scale is applied, 424 combinatorialvariants are possible. In practice, MBC methods yield a set ofsequences, each of which originated from a primordial single-strandedDNA template. Unique tag-identified duplicates are compared such thatmutations are scored only when the same mutation is preset in multipleunique tags. (If a NextSeq 500 generates ˜400×106 reads, this indicateseach read could be individually labelled by 1 of ˜700,000 uniquesequencing identifiers. (If N8, each read could be individually labelledby ˜10 unique MBC). In summary, it is calculated that this dualmolecular barcoding approach represents a 10 million-fold errorimprovement over the 3.8×10-3 error value obtained using standardIllumina methods (Schmitt et al., (2012) Proceedings of the NationalAcademy of Sciences of the United States of America, 109, 14508-14513.The same error correction can be achieved by ordering oligonucleotidesbearing the random N sequence stretch manufactured using premixedequimolar concentrations of G, A T and C phosphoramidites duringoligonucleotide manufacture. There are multiple examples of NGS errorreduction (US 20120108467) approaches in the patent literature includingmolecular counting (WO 2007087312) and stochastic labelling (US20130116130) respectively.

Instructions for using the kit to perform one or more methods of thedisclosure can be provided, and can be provided in any fixed medium. Theinstructions may be located inside or outside a container or housing,and/or may be printed on the interior or exterior of any surfacethereof. A kit may be in multiplex form for concurrently detectingand/or quantitating one or more different target polynucleotidesrepresenting the expressed target molecules.

Devices useful for performing methods of the disclosure are alsoprovided. The devices can comprise means for amplifying and/orsequencing a genome or target molecules thereof, for example componentsfor performing one or more methods of nucleic acid extraction,amplification, and/or detection. Such components may include one or moreof an amplification chamber (for example a thermal cycler), a platereader, a spectrophotometer, capillary electrophoresis apparatus, a chipreader, and or robotic sample handling components. These componentsultimately can obtain data that reflects the expression level of thetarget molecules used in the assay being employed.

The devices may include an excitation and/or a detection means. Anyinstrument that provides a wavelength that can excite a species ofinterest and is shorter than the emission wavelength(s) to be detectedcan be used for excitation. Commercially available devices can providesuitable excitation wavelengths as well as suitable detection component.

Exemplary excitation sources include a broadband UV light source such asa deuterium lamp with an appropriate filter, the output of a white lightsource such as a xenon lamp or a deuterium lamp after passing through amonochromator to extract out the desired wavelength(s), a continuouswave (cw) gas laser, a solid state diode laser, or any of the pulsedlasers. Emitted light can be detected through any suitable device ortechnique; many suitable approaches are known in the art. For example, afluorimeter or spectrophotometer may be used to detect whether the testsample emits light of a wavelength characteristic of a label used in anassay.

The devices typically comprise a means for identifying a given sample,and of linking the results obtained to that sample. Such means caninclude manual labels, barcodes, and other indicators which can belinked to a sample vessel, and/or may optionally be included in thesample itself, for example where an encoded particle is added to thesample. The results may be linked to the sample, for example in acomputer memory that contains a sample designation and a record ofexpression levels obtained from the sample. Linkage of the results tothe sample can also include a linkage to a particular sample receptaclein the device, which is also linked to the sample identity.

The devices also comprise a means for correlating the presence orabsence of the target molecules being studied with a prognosis ofdisease outcome. Such means may comprise one or more of a variety ofcorrelative techniques, including lookup tables, algorithms,multivariate models, and linear or nonlinear combinations of expressionmodels or algorithms. The data may be converted to one or morelikelihood scores, reflecting likelihood that the patient providing thesample may exhibit a particular disease outcome. The models and/oralgorithms can be provided in machine readable format and can optionallyfurther designate a treatment modality for a patient or class ofpatients.

The device also comprises output means for outputting the diseasestatus, prognosis and/or a treatment modality. Such output means cantake any form which transmits the results to a patient and/or ahealthcare provider, and may include a monitor, a printed format, orboth. The device may use a computer system for performing one or more ofthe steps provided.

The methods disclosed herein may also comprise the transmission ofdata/information. For example, data/information derived from thedetection and/or quantification of the target may be transmitted toanother device and/or instrument. In some instances, the informationobtained from an algorithm may also be transmitted to another deviceand/or instrument. Transmission of the data/information may comprise thetransfer of data/information from a first source to a second source. Thefirst and second sources may be in the same approximate location (e.g.,within the same room, building, block, campus). Alternatively, first andsecond sources may be in multiple locations (e.g., multiple cities,states, countries, continents, etc).

Transmission of the data/information may comprise digital transmissionor analog transmission. Digital transmission may comprise the physicaltransfer of data (a digital bit stream) over a point-to-point orpoint-to-multipoint communication channel. Examples of such channels arecopper wires, optical fibres, wireless communication channels, andstorage media. The data may be represented as an electromagnetic signal,such as an electrical voltage, radiowave, microwave, or infrared signal.

Analog transmission may comprise the transfer of a continuously varyinganalog signal. The messages can either be represented by a sequence ofpulses by means of a line code (baseband transmission), or by a limitedset of continuously varying wave forms (passband transmission), using adigital modulation method. The passband modulation and correspondingdemodulation (also known as detection) can be carried out by modemequipment. According to the most common definition of digital signal,both baseband and passband signals representing bit-streams areconsidered as digital transmission, while an alternative definition onlyconsiders the baseband signal as digital, and passband transmission ofdigital data as a form of digital-to-analog conversion.

EXPERIMENTAL Example 1

This example describes an exemplary whole genome amplification methodperformed in tubes. FIG. 1 shows an overview of the amplificationmethods.

Three oligonucleotides (described in Table 1) known as: (MALBACN53G);(MALBACN543T) and (MALBAC 27) were ordered from Integrated DNATechnology.

TABLE 1 Oligonucleotides 1. Name Sequence MALBACN53GGTGAGTGATGGTTGAGGTAGTGTGGAGNNNNNG*G* G (SEQ ID NO: 1) MALBACN543TGTGAGTGATGGTTGAGGTAGTGTGGAGNNNNNT*T* T (SEQ ID NO: 2) MALBAC 27GTGAGTGATGGTTGAGGTAGTGTGGAG  (SEQ ID NO: 3) Note: “*” indicates asulphur modified phosphodiester bond (phosphorothioate), and “N”indicates an equimolar concentration of G, A, T and C were introducedinto oligonucleotides during manufacture.

The MALBAC 27 sequence is present on both ends of the resultant WGAamplicon. Phi29 polymerase, buffer and BSA were purchased from NEB;dNTPs and PCR components were purchased from Roche. MALBACN53G andMALBACN53T were employed at 0.32 and 0.25 μM, respectively. DNA 6, 18 or60 pg., (mass equivalent to 1, 3 and 10 human genomes respectively) wasannealed in 10 μL. A second 10 μL aliquot containing WGA reactioncomponents at 2× concentration (see FIG. 1 for components) was added(stacked) on top of the first 10 μL reaction. A third 10 μL aliquotpermitting PCR to occur (Hi Fi DNA polymerase and Buffer, Roche) wasadded. The final volume consisting of 3×10 μL aliquot dispenses wasformed. An NTC consisting of passaging water through the process wasperformed. Base-level and limited Phi29 amplification was performed at30° C. for 20 min. followed by 3 hours at 40° C. to minimize TIDA(Alsmadi et al., (2009) BMC Res Notes, 2, 48), and decrease ampliconlength, the propensity for undesired intra-primer extension andrecurrent preamplification from existing amplicons. 40° C. was chosen toboth limit processivity while not inhibiting any amplicon panhandleeffects (Jones, D. H. (1995) PCR Methods Appl, 4, S195-201; Jones, D. H.and Winistorfer, S. C. (1993) PCR Methods Appl, 2, 197-203; Robinson etal., (2006) Genes Chromosomes Cancer, 45, 740-753). PCR was performedusing a single primer (US Pat. App. No. 20120100549) to amplify fromboth ends.

Reaction products were purified from low MW reaction products andreaction components using AMPure Beads (Beckman) to 0.8× concentration(twice). The same DNA samples were amplified using the MALBAC protocolas per manufacturer's instructions (Yikon Genomics). All samples weretreated by standard Nextera XT tagmentation processes (Illumina).Tagmentation reactions were sized on a Bioanalyzer Hi Sensitivity Chip(Agilent) and sequenced via a single MiSeq 250 bp sequencing run(SeqMatic Corporation, Fremont).

FIGS. 2a and 2b illustrate bioanalyzer sizing traces generated usingMALBAC and the compositions and methods described herein, respectively.The MALBAC procedure generated a greater proportion of large fragmentedhigher MW material and NTC amplicons that were not observed using thecompositions and methods described herein. In this example, NTCamplicons were observed in the MALBAC Nextera prep. NTC amplificationwas not observed in material prepared using the compositions and methodof embodiments of the present disclosure.

WGA in nL Volume Reaction Vessels:

A WGA method was performed in Wafergen in nL volume reaction vessels. 50nL volumes containing “1” or “10” U937 cells; 6 or 60 pg of U937 gDNA(prepared from bulk U937 tissue culture cells using Qiagen DNApreparation column), or a NTC were dispensed into 108 wells of a5184-well “deep well” (350 nL) chip using a Wafergen Multiple SampleNano Dispenser (MSND). All samples were dispensed in 10% glycerol. 5separate chips were examined in this manner. Chips were sealed withoptical grade sealant covers and frozen at −80 C. At a later time (7days later) chips were thawed at room temperature for 10 mins. TheMALBAC primers (100 nL, described earlier) were added in the presence of40 ug Proteinase K (Ambion). A sealant cover was added and chips wereincubated at 50 C/25 mins, 95 C/20 mins; 37 C 5 mins followed by a 4 Chold. The Phi29 amplification step was performed by addition of 50 nL(scaled using concentrations described in FIG. 1). The Phi29amplification step was followed with a PCR reaction also performed asper FIG. 1 but with the modification that EvaGreen DNA binding dye(Biotium) was added to 1× concentration.

PCR thermal cycling was performed using a Wafergen Smart Cycler.Reactions in all 5184 wells were monitored for PCR amplicon productionvia Cq and Tm measurement. Data was deconvoluted from the 350 nL chipsusing Wafergen qPCR software in combination with Excel. Wafergen qPCRsoftware amplification signals cutoffs were set to the highest leniencyin an attempt to detect as many NTC signals as possible. The totalvolume in each reaction was approximately 250 nL. FIG. 6 provides inchip Cq and Tm heat maps for 6 and 60 pg of gDNA positive control (1 and10 cell equivalents) and individual 1 and 10 cell samples as dispensedin the chip. A NTC demonstrates the back ground level of amplificationis very low and easily distinguished form test and positive controlsamples.

The contents of each chip were collected by centrifugation. On chipamplicons were Nextera treated and prepared for sequencing using anIllumina MiSeq sequencer. FIGS. 7A-B and 8A-B demonstrate sequencedbases covered/chromosome/total bases covered for U937 gDNA (positivecontrol) and 1 and 10 cell samples respectively. In general there isclose alignment between expected (NCBI reference) and U937 (cancer cellline) chromosome read distribution. The chromosome level distributiondifference between expected and observed reads may represent expectedgenomic differences between the cancerous U937 cell line and the NCBIreference human genome.

Process Sequencing Results:

Data reads across chromosomes (Chr.) was examined for both the systemsand methods described herein and MALBAC using the same DNA sample(XXXX.arr(X)x4,(Y)x0) was obtained from the Coriell institute. Separatesamples were barcoded and prepared using Nextera and sequenced using asequencing MiSeq instrument. The FASTQ files from the same sequencingrun were aligned to the human genome. In brief, it is known that humanChr. are named in order of their length with Chr. 1 being longer thanChr. 2 etc. The lengths of each Chr. were obtained from NCBI. FIGS. 3A-Cillustrate the method at each concentration (6, 18 and 60 pg.), a plotof mapped reads per Chr. versus Chr. number (surrogate for length) wasdrawn. FIGS. 4A-C compares sequenced counts per chromosome for bothmethods (6, 18 and 60 pg.). The reference line plots the referenceunamplified counts for an unamplified human genome on a per Chr. basis.The black line indicates how well each method input matches thatreference. The R² coefficients are more linear between 6-to-60 pg.inputs than the equivalent MALBAC R².

Across all sample inputs the R² correlation coefficient between expectedand observed slopes decreased with increased input. This indicates thata common reagent depletion effect may be occurring as the sample inputconcentration increases. This “reagent depletion” effect was lesspronounced for the compositions and methods of embodiments of thepresent disclosure at all concentrations than for the MALBAC procedure.In broad summary, the method described herein generates equivalent dataas MALBAC, using a higher proofreading polymerase and a simplifiedprocess.

In some embodiments, each sample in a smart chip engages with a uniquepairing of WGA priming oligonucleotides that bear SBC pre-synthesized inthe primer sequences. A cost effective mode of achieving 5184 uniquecombinations of P5 and P7 bearing SBC in a 72×72 chip is to add(preprint) P5 engageable oligos bearing a common SBC to all wells incolumn 1. Column 2 receives a separate common SBC with P5 engageableends. This will be continued to column 72. Each column will contain 1 of72 column unique SBC with P5 engageable ends. In the same manner, eachrow in the chip will receive a P7 engageable oligo with SBC 73 in row 1,SBC 74 in row 2 etc. to row 72. This approach produces an intersectionof P5 and P7 oligos with 2 separate SBC oligos. Only 144 separate 72columns×72 row oligos need to be designed to yield 5184 unique SBCbarcode combinations. It will be obvious to those knowledgeable in theart that the engageable tag sequence can be a variety of sequenceidentities including Ion Torrent PGM sequencing adaptors (A and P1) orother tags permitting further amplification such as T7 DNA polymerasebinding sites (Zhu, B. (2014) Frontiers in microbiology, 5, 181; VanGelder et al., (1990) Proceedings of the National Academy of Sciences ofthe United States of America, 87, 1663-1667) for subsequent microarrayor a variety of other genetic analyses.

Creating the SBC matrix before sample is added (preprinted) decreasesthe number of stacking biochemistry reactions customers must perform.This makes the system more robust. It decreases R & D investigativeinvestment costs. It also enables poor oligo syntheses to be rapidlyidentified for QC purposes. Unique pairings of SBC can be added in the1× or 2× reaction mixtures described in FIG. 1. FIG. 5A illustratesthose primers of the general form: (SEQ ID NO: 3)GTGAGTGATGGTTGAGGTAGTGTGGAG (SBC 6-10mer)(MBC; N6-N10)(N5-KKK), whereK=G or T. FIG. 5B illustrates an exemplary in line dual SBC and MBCprimer-amplicon configuration. The 27 mer MALBAC or other tag sequenceis present on the end of all WGA and PCR amplicons to incorporate thissequence as a component of a custom primer. This avoids sequencingunwanted tag sequence. FIG. 5B shows elongating the MALBAC sequence by16 padding bases to bring its Tm to ˜65 C (in a Mg2+ free milieu). FIG.5B illustrates the 16 bases of padding sequence 5′ of the MALBACsequence plus ˜5 bases of the MALBAC sequence can be used to attachIllumina P5 and P7 flow cells sequences in bulk, off chip.

The elongated MALBAC primer is manually added to ports 12 and 14 of aMiSeq cartridge and its equivalent cartridge positions used in Next Seqand HiSeq systems. This primer binds upstream of a sample barcode andincludes the SBC and MBC sequence within read 1. The reverse complementof the elongated MALBAC primer (Read 2) is added to ports 13 in a MiSeqand its equivalent cartridge positions used in Next Seq and HiSeqsystems. This primer binds upstream of a sample barcode and includes SBCand MBC sequence distinct from read 2. SBC and MBC are bioinformaticallyde-multiplexed by end users.

All publications and patents mentioned in the present application areherein incorporated by reference. Various modification and variation ofthe described methods and compositions of the disclosure will beapparent to those skilled in the art without departing from the scopeand spirit of the disclosure. Although the disclosure has been describedin connection with specific preferred embodiments, it should beunderstood that the disclosure as claimed should not be unduly limitedto such specific embodiments. Indeed, various modifications of thedescribed modes for carrying out the disclosure that are obvious tothose skilled in the relevant fields are intended to be within the scopeof the following claims.

We claim:
 1. A method of whole gene amplification (WGA), the methodcomprising: a) combining: a sample of genomic DNA template; a phi29polymerase; nucleic acid primers; and dNTPs; into a reaction mixtureunder conditions sufficient for genomic DNA template-directed extensionof the nucleic acid primers; and b) amplifying the extended nucleic acidprimers in the reaction mixture with a thermostable DNA polymerase togenerate amplified whole genome DNA, wherein said combining andamplifying are performed in a single vessel.
 2. The method of claim 1,wherein said sample of genomic DNA is a single cell sample.
 3. Themethod of claim 1, wherein said sample of genomic DNA is a plurality ofcells.
 4. The method of claim 1, further comprising the step ofdetecting one or more nucleic acid variants in said amplified wholegenome DNA.
 5. The method of claim 1, wherein said method furthercomprises the step of performing a nucleic acid sequencing assay on saidamplified whole genome DNA.
 6. The method of claim 1, wherein saidamplification is conducted in a volume of 35 to 500 nL.
 7. The method ofclaim 1, wherein at least a subset of said primers are specific for anucleic acid target of interest.
 8. The method of claim 1, wherein atleast a subset of said primers comprises a sample barcode, molecularbarcode, label, or tag sequence.
 9. The method of claim 1, wherein saidvessel is a well of a microfluidic chip comprising a plurality of wells.10. The method of claim 9, wherein each of said wells comprises adistinct nucleic acid primer.